Abstract
Background: Fibulin-4, a matricellular protein, plays a major role in the development of cardiovascular extracellular matrix, since Fbln4 KO mice die in the neonatal period because of arterial rupture, and smooth muscle-specific Fbln4 CKO mice exhibit ascending aortic aneurysm. However, its molecular function remains elusive. We hypothesized that fibulin-4 is required for the activity of lysyl oxidase (LOX), a key enzyme for cross-linking collagens and elastin, because the phenotypes of Lox KO mice were similar to those of Fbln4 KO mice.
Methods: Collagen and elastin crosslink products were measured in WT and Fbln4 KO mice tissues. LOX in WT and Fbln4 KO MEFs was analyzed by Western blot. LOX was purified from WT and Fbln4 KO MEF, and the activity and posttranslational modification of purified LOX was analyzed.
Results: Aortic tissues from Fbln4 KO mice and Lox KO mice showed greatly reduced collagen and elastin crosslinks. LOX produced by Fbln4 KO cells migrates slowly in SDS-PAGE, and had much lower activity than that produced by WT cells. These were reversed by Fibulin-4 overexpression or by the addition of recombinant Fibulin-4. Nitro blue tetrazolium staining and mass-spectrometric analysis of purified LOX revealed that most of LOX produced by Fbln4 KO cells does not contain lysine tyrosyl quinone (LTQ), an intramolecular crosslink that works as a cofactor of LOX. LTQ formation requires copper, but LOX produced in the absence of Fibulin-4 contained much lower copper than that produced in the presence of Fibulin-4. These data indicate that Fibulin-4 is required for copper incorporation into LOX, a necessary step for formation of LTQ that is essential for LOX activity.
